Chromatography (PC, CC).pptxpharmaceutical analysis

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1. Theory, instrumentation, and application of paper and column chromatography DEPT OF PHARMACEUTICAL ANALYSIS

2. CHROMATOGRAPHY

3. Mikhail Semyonovich Tswett (1872-1919 ) In the beginning of 19 th century ( 1903 ) a Russian scientist ‘ M.S . Tswett ’ while working on plant extracts encountered coloured bands that moved down the column. He named chromatography to this technique. Invention of Chromatography he is considered as Father of chromatography .

4. What is Chromatography? Chromatography is a technique for separating mixtures into their components in order to purify, identify, and/or quantify the mixture or components. Separate Purify Identify Quantify Components Mixture

5. Advantages Components from a complex mixture can separate. A small amount of sample (milli, micro, and ng) can be detected by this chromatography. It is a rapid and precise method of separation. Very few sample volume/quantity is required for analysis. It works on a broad range of samples. In some chromatography techniques, it is possible to separate different components of a complex mixture. Continuous operation possible on a large scale The separation of components can be achieved in different methods. disadvantages of Chromatography: The chromatography equipment can only be operated by a trained person. Chromatography instruments are expensive. An error occurs due to the overloading of the samples. Chromatography equipment must be handled with care because of these parts are expensive and sensitive. Some of the chromatography techniques require more solvent to separate the analytes. Some of the chromatography methods require high power consumption. High operational pressure may be required to achieve efficient separation.

6. By stationary phase usage Adsorption: TLC, Column. Partition: Paper, HPLC, GC. Ion-Exchange. Size Exclusion. Classification of Chromatography

7. Gas chromatography-mass spectrometry (GC-MS) Gas chromatography-infra red spectroscopy (GC- IR) Liquid chromatography-mass spectrometry (LC-MS): Quadrupole and TOF Liquid chromatography-nuclear magnetic resonance spectroscopy (LC-NMR) Capillary electrophoresis- mass spectrometry (CE-MS) Super critical fluid chromatography- mass spectrometry (SFC-MS) Liquid chromatography-inductively coupled plasma mass spectrometry (LC-ICP-MS) Gas Chromatography (GC) Column chromatography (LC) Paper chromatography (PC) Thin layer chromatography (TLC) High performance thin layer chromatography (HPTLC) High performance liquid chromatography(HPLC) Supercritical fluid chromatography (SFC) Size-exclusion chromatography(SEC/GPC) Ion exchange chromatography (IEC) Affinity chromatography (AC) Counter current chromatography (CCC) Flash chromatography (FC) Ultra pressure liquid chromatography (UPLC) Simulated moving bed chromatography (SMBC) Different forms of Modern chromatography Hyphenated Techniques Separation Techniques

8. General Principles of Chromatography Separation of mixtures by distribution between a stationary phase and a mobile phase. A stationary phase ( absorbent ) phase the material on which the separation takes place. can be solid, gel, or liquid . Also called matrix, resin, or beads. The mobile phase is the solvent transports the sample and it is usually a liquid, but may also be a gas . Also called eluting buffer The compounds to be separated are considered solutes

9. Adsorption chromatography is probably one of the oldest types of chromatography around. It utilizes a mobile liquid or gaseous phase that is adsorbed onto the surface of a stationary solid phase . The equilibration between the mobile and stationary phase accounts for the separation of different solutes. 1.Adsorption Chromatography :

10. This form of chromatography is based on a thin film formed on the surface of a solid support by a liquid stationary phase. Solute equilibrates between the mobile phase and the stationary liquid. 2.Partition Chromatography :

11. In this type of chromatography, the use of a resin (the stationary solid phase) is used to covalently attach anions or cations onto it. Solute ions of the opposite charge in the mobile liquid phase are attracted to the resin by electrostatic forces. 3.Ion Exchange Chromatography :

12. Also known as gel permeation or gel filtration , this type of chromatography lacks an attractive interaction between the stationary phase and solute. The liquid or gaseous phase passes through a porous gel which separates the molecules according to its size . The pores are normally small and exclude the larger solute molecules , but allows smaller molecules to enter the gel, causing them to flow through a larger volume . This causes the larger molecules to pass through the column at a faster rate than the smaller ones . 4.Molecular Exclusion Chromatography :

13. STATIONARY PHASE Type of chromatography Material Paper chromatography Filter paper, cellulose Thin Layer Chromatography Silica gel, alumina, Gas chromatography Squalene , apezion , carbowax M High Performance Liquid Chromatography C-8, C-18,

14. Type of chromatography Solvent Paper chromatography Organic solvents Thin Layer Chromatography Hexane, ether petroleum, alcohol. Gas chromatography He, Ar , N 2 High Performance Liquid Chromatography Water, Acetonitrile, Methanol Cyclohexane, n-hexane, carbon tetrachloride, ethanol, MOBILE PHAS E

15. Chromatograph – Equipment used for separation. EX. Gas chromatography or Liquid chromatography Eluent - Mobile phase/solvent that carries the analyte. Eluate - Mobile phase leaving the column along with Analyte . Stationary phase - Immobilized phase: Immobilized on the support particles or on the inner wall of the column tubing. Example: Silica layer - Thin Layer Chromatography CHROMATOGRAPHY TERMS

16. Mobile phase Moves in a definite direction. Liquid (LC: Water, Organic solvents), Gas (GC). Flows through the column carry analyte. The mobile phase moves through the chromatography column (the stationary phase) where the sample interacts with the stationary phase and is separated. Retention time (Rt): The time takes for a particular analyte to pass through the system (from the column inlet to the detector) under set conditions ( HPLC ). Retention factor (Rf): Ratio between solute and Solvent front ( PC ) Sample (Analyte) : Substance analyzed in chromatography. Solvent : Any substance capable of solubilizing another substance. The SEPARATION is based on the Partitioning: PC, HPLC, GLC Adsorption: TLC. CC between the mobile and stationary phase.

17. Chromatogram: Visual output of the chromatographic equipment . Separation/Resolution - Different peaks on the chromatogram correspond to different components of the separated mixture.

18. PAPER CHROMATOGRAPHY

19. Paper chromatography is an analytical method that is used to separate colored chemicals or substances, especially pigments. This is useful for separating complex mixtures of compounds having similar polarity, for example, amino acids. If a filter paper is used, it should be of a high quality paper . The mobile phase is developing solutions that can travel up to the stationary phase carrying the sample along with it. Definition:

20. The principle involved is partition chromatography where in the substances are distributed or partitioned between two liquid phases. One phase is the water which is held in the pores of filter paper (stationary phase) used and the other phase is that of the mobile phase which moves over the paper. The compounds in the mixture get separated due to differences in their affinity towards water (in the stationary phase) and mobile phase solvents during the movement of the mobile phase under the capillary action of pores in the paper . Principle : the process of a  liquid  flowing in a narrow space without the assistance of, any external forces like  gravity .

21. Descending paper chromatography. Ascending paper chromatography. Ascending and descending paper chromatography. Radial paper chromatography or circular chromatography. Two dimensional paper chromatography. Types of paper chromatography

22. 1.Descending Paper Chromatography -In this type, development of the chromatogram is done by allowing the solvent to travel down the paper is called Descending Chromatography. Here, the mobile phase is present in the upper portion. 2. Ascending Paper Chromatography -Here the solvent travel upward direction of the Chromatographic paper. Both the Descending and Ascending Paper Chromatography are used for separation of Organic and Inorganic substances.

23. Ascending paper chromatography Descending paper chromatography

24. 3. Ascending-Descending Paper Chromatography -It is the hybrid of both the above techniques. The upper part of the Ascending chromatography can be folded over a rod and allowing the paper to become descending after crossing the rod. 4. Radial Paper Chromatography -It is also called as Circular chromatography. Here a circular filter paper is taken and the sample is spotted at the center of the paper . After drying the spot the filter paper is tied horizontally on a Petri dish containing solvent. So that Wick of the paper is dipped inside the solvent. The solvent rises through the wick and the component get separated in form of concentrate circular zone.

25. Ascending and descending paper chromatography Radial chromatography

26. 6. Two-Dimensional Paper Chromatography -In this technique a square or rectangular paper is used. Here the sample is applied to one of the corners and development is performed at right angle to the direction of first run.

27. The retention factor ( R ƒ ) may be defined as the ratio of the distance traveled by the substance to the distance traveled by the solvent. R ƒ  values are usually expressed as a fraction of two decimal places. If R ƒ  value of a solution is zero , the solute remains in the stationary phase and thus it is immobile. If R ƒ  value = 1 then the solute has no affinity for the stationary phase and travels with the solvent front. To calculate the R ƒ  value, take the distance traveled by the substance divided by the distance traveled by the solvent (as mentioned earlier in terms of ratios). Retention factor value (R ƒ ):

28. R ƒ = distance traveled by the substance/Solute = 4 = 0.4 distance traveled by the solvent 10 Calculation:

29. Selection of suitable type of development Selection of suitable filter paper (Whatman) Preparation of sample Spotting of sample on the paper Development of chromatogram Drying of the paper and detection of the compounds Experimental procedure (Steps):

30. The experimental method involves 1.Selection of suitable type of development:  This depends on complexity of the mixture, solvent, paper etc. But in general ascending type or radial type of chromatography are used as they are easy to perform, handle, less time consuming and also give chromatogram faster. 2.Selection of suitable filter paper : Filter paper is selected based on pore size, quality of the sample to be separated and also mode of development. Experimental procedure

31. 3.Preparation of sample:  Preparation of sample involves dissolution of sample in suitable solvent used in making mobile phase. The solvent used should be inert with the sample under analysis. 4.Spotting of sample on the paper. Samples are to be spotted at proper position on the paper using preferably a capillary tube.

32. 5.Development of chromatogram:  Sample spotted paper is subjected to development by immersing it in the mobile phase. The mobile phase moves over the sample on the paper under the capillary action of paper . 6.Drying of the paper and detection of the compounds : Once the development of chromatogram is over, The paper is held carefully at the borders so as to avoid touching the sample spots and dried using an air drier. Sometimes the detecting solution is sprayed in the developed paper and dried to identify the sample chromatogram spots.

33. Visualizing Agent :- After development of chromatogram the spot should be visualized. It can done by two Method A . Nonspecific methods B. Specific methods A.  Nonspecific methods  :- where the number of spot can be detected but not the exact nature or type of compound Example  1. Iodine chamber method :- where brown or Amber spots are observed when the TLC plates are kept in a tank with few iodine crystal at the bottom. 2. UV chamber for fluorescent compound :- when compound are viewed under UV chamber at 254 nm or at 365 nm fluorescent compound can be detected. B.  Specific methods  :- Specific spray reagents or detecting agent or visualising agent are used to find out the nature of compound. Example Ferric chloride – for phenolic compound and tannins Ninhydrin in Acetone – for amino acids. Dragendroff reagents – for alkaloids DETECTING OR VISUALISING AGENTS

34. Paper chromatography is specially used for the separation of mixtures having polar and nonpolar compounds in plant mixtures. For separation of Proteins, peptides, NA, amino acids. It is used to determine organic compounds, and biochemicals in biological samples (Plasma, urine). In the pharma sector for separation of determination of drugs. Applications:

35. Prepare solution of 0.1g/100ml each of valine, threonine, glycine, crystine and isoleucine. 8×10 inch sheet of Whatmann filter paper No.1 and place six equally spaced small circles with a pencil, 0.5 inch from the bottom. Label the paper at the top with the name of each amino acid and label the sixth unknown(for an unknown mixture of acids). Place small drops of an amino acid on one circle and dry it carefully over the plate between each drop application. Now curl or roll the paper sheet into cylindrical form and put it into the jar or beaker containing a mixture of 95% ethyl alcohol and 5% water to a depth of about 0.25 inch and cover the beaker with a lid or watch glass. That the paper does not touch the sides of the beaker. The top and bottom of the paper are stapled. Ex: Separation of amino acids by paper chromatography

36. Now allow the eluting agent to rise about 6 inch. Its takes about 3 hours. Then remove the paper from the beaker and immediately mark the solvent front position with the help of pencil. The chromatogram is dried and then sprayed with 0.25% ninhydrin solution in water saturated with butyl alcohol. Now allow chromatogram to dry in an oven for few minutes at about 100-105 c until color spots develop. The different amino acids appear as blue like spots. Calculate the R ƒ values of the amino acids.

37. COLUMN CHROMATOGRAPHY

38. Column chromatography is basically a type of adsorption chromatography techniques. Here the separation of components depends upon the extent of adsorption to stationary phase. Here the stationary phase is a solid material packed in a vertical column made of glass or metal and the mobile phase is either gas or a liquid. Definition:

39. When a mixture of mobile phase and sample to be separated are introduced from top of the column, the individual components of mixture move with different rates. Those with lower adsorption/affinity to the stationary phase move faster and eluted out first while those with greater adsorption/affinity move or travel slower and get eluted out last. The solute molecules adsorb to the column in a reversible manner. The rate of the movement of the components is given as follows Principle:

40. Adsorbents: The usual adsorbents employed in column chromatography are silica, alumina, calcium carbonate, calcium phosphate, magnesia, starch , etc., Alumina is generally suitable for chromatography of less polar compounds. Silica gel gives good results with compounds containing polar functional groups. Experimental aspects:

41. Particles should be spherical in shape & uniform in size . Mechanical stability must be high. They shouldn’t react chemically . It should be useful for separating for wide variety of compounds. It should be freely available & inexpensive (The particle size of the commercially available grade is in the range 50 – 200 µm .) Adsorbent in column chromatography should meet following criteria:

42. Success of chromatography depends upon proper selection of Stationary phase, it depends on the following: Removal of impurities. Number of components to be separated. Length of the column used. Affinity differences between components. Quantity of adsorbent used. Selection of stationary phase:

43. TYPES OF ADSORBENTS

44. COLUMN CHROMATOGRAPHY Experimental aspects of column chromatography: Adsorbents: The usual adsorbents employed in column chromatography are silica, alumina, calcium carbonate, calcium phosphate, magnesia, starch, etc., Alumina is generally suitable for chromatography of less polar compounds. Silica gel gives good results with compounds containing polar functional groups.

45. Silica gel Silica gel is a granular, porous form of silica made synthetically from sodium silicate . Despite the name, silica gel is a solid. Silica gel is used in chromatography as a stationary phase. In this application, due to silica gel's polarity, non-polar components tend to elute before more polar ones, hence the name normal phase chromatography . However, when hydrophobic groups (such as C18 groups) are attached to the silica gel then polar components elute first and the method is referred to as reverse phase chromatography . Silica gel is also applied to aluminum or plastic sheets for thin layer chromatography

46. Difference between Normal Phase & Reverse Phase Chromatography Normal Phase Chromatography Reverse Phase Chromatography  It uses a polar stationary phase and a non-polar (low Polarity Solvents) mobile phase.  Non-polar compounds elute faster than polar compounds. When we increase polarity of mobile phase elution time will increase. It can not be reused / reproducible Mobile phase are non polar i.e. IPA, hexane, dichloromethane, chloroform, ethyl ether, and isopropyl alcohol (IPA). It uses a non polar stationary phase and a polar mobile phase. Polar compounds elute faster than non polar compounds. When we increase polarity of mobile phase elution time will decrease. It Can reused / reproducible Mobile phase are polar compounds such as water, acetonitrile, methanol

47. They act as solvent , developer & eluent . The function of a mobile phase are: As developing agent. To introduce the mixture into the column – as solvent. To developing agent. To remove pure components out of the column – as eluent. Selection of mobile phase: The choice of the solvent is depend on the solubility characteristics of the mixture. The solvents should also have sufficiently low boiling point to permit the ready recovery of eluted material. However, polarity as seen the most important factor in adsorption chromatography. It can be used in either pure form or as mixture of solvents

48. The main function of all the columns is to support the stationary phase. The material of the column is mostly good quality neutral glass since it shouldn’t be affected by solvents. An ordinary burette can also be used as column for separation. Column dimensions - length & diameter ratio ( 10:1,30:1 or 100:1cm ) Various accessories are attached to the top and bottom of the column for maintenance of the elution process. A Column characteristics:

49. The length of the column depends upon: Number of compounds to be separated. Type of adsorbent used. Quantity of the sample . Affinity of compounds towards the adsorbent used. Better separation will be obtained with a long narrow column than short thick column because number of plates will be more.

50. It consists of a glass tube with bottom portion of the column – packed with glass wool/cotton wool or may contain asbestos pad. Above which adsorbent is packed. After packing a paper disc kept on the top, so that the adsorbent layer is not disturbed during the introduction of sample or mobile phase. Demerit: Air bubbles are entrapped between Mobile phase. Preparation of the column: There are two types of preparing/packing the column, they are: Dry packing / dry filling. Wet packing / wet filling .

51. Column chromatography Dry Packing Technique : The Stationary phase cracks appear in the adsorbent layer. After filling tapping can be done to remove void spaces. Column chromatography wet packing technique: Ideal & common technique. The material is slurred with solvent and generally added to the column in portions. Stationary phase settles uniformly and no crack in the column of adsorbent. Solid settle down while the solvent remain upward. This solvent is removed then again cotton plug is placed.

52. Dry Packing Technique Adsorbent is packed in the column in dry form Fill the solvent, till equilibrium is reached DEMERIT: Air bubbles are entrapped b/w M.P & S.P→ cracks appear in the adsorbent layer. After filling tapping can be done to remove void spaces.

53. Wet Packing Technique » ideal & common technique The material is slurried with solvent and generally added to the column in portions. ◊ S.P settles uniformly & no crack in the column of adsorbent. » solid settle down while the solvent remain upward. » this solvent is removed then again cotton plug is placed.

54. Introduction of the Sample The sample which is usually a mixture of components is dissolved in minimum quantity of the mobile phase. The entire sample is introduced into the column at once and get adsorbed on the top portion of the column. From this zone, individual sample can be separated by a process of elution.

55. Wet Packing Technique

56. By elution technique, the individual components are separated out from the column. The two techniques are: 1.Isocratic elution technique : In this elution technique , same solvent composition or solvent of same polarity is used throughout the process of separation . Example: chloroform only 2.Gradient elution technique : Solvents of gradually increases polarity or increases elution strength are used during the process of separation . E.g. Initially benzene, then chloroform, then ethyl acetate then chloroform Development/elution technique:

57. If the compounds separated in a column chromatography procedure are colored , the progress of the separation can simply be monitored visually . If the compounds to be isolated from column chromatography are colorless . In this case, small fractions of the eluent are collected sequentially in labeled tubes and the composition of each fraction is analyzed by TLC . Detection of components:

58. Different components are separate as column progresses. Fractions can be collected in test tubes, vials, beakers, or Erlenmeyer flasks. Eluting the sample:

59. Analyze the fractions by thin-layer chromatography. Analyzing the fraction:

60. Dimension of the column : column efficiency has been improved by increasing length/width ratio of the column. Particle size of column packing : separation to be improved by decreasing the particle size of the adsorbent. Activity of the adsorbent . Temperature of the column : The speed of the elution increases at higher temperatures. Packing of the column . Quality of solvents : solvents having low viscosities is giving better results. Factors affecting column efficiency:

61. Procedure: The stationary phase material is suitably moistened with mobile phase and packed sufficiently in the column with a cotton or asbestos pad at the bottom. The extract material or sample to be separated is placed on the top of packed stationary phase with a second cotton or asbestos pad in between. The mobile phase is poured into the column over the sample. A collecting beaker is placed at the bottom of column near the end to collect the elute.

62. Separate active principle from plant materials. In separation of compounds after organic synthesis to obtain desired molecule. To separate or purify natural compound mixtures like alkaloids, glycosides. Separation of mixture of compounds. Purification process. Applications:

63. Isolation of active constituents. Estimation of drugs in formulation. Isolation of active constituents. Determination of primary and secondary glycosides in digitalis leaf. Separation of diastereomers.

64. Stationary phase- slurry of 25g of alumina(neutral 100-200 mesh) in ethyl alcohol. Sample - 5mg each of methylene blue in 5ml of ethyl alcohol. Allow the eluent to drain in the column to within 1mm of the top of the alumina. Mobile phase- ethyl alcohol. Elute the methylene blue from the column, collect the eluent in 5ml aliquots in vials. After all the methylene blue has been collected, rate the relative concentration of each vial from 1to 10 from the depth of blue. Now plot the relative concentration of each vial against the tube number and obtain the chromatogram. Repeat the experiment using 50% water, 50% ethyl alcohol and 100% water eluents. Compare the data for the three eluting agents. Separation of methylene blue by column chromatography:

65. Advantages: Any type of mixture can be separated. Any quantity of mixture can be separated. Wider choice of Mobile Phase. Automation is possible. Disadvantages: Time consuming. More amount of Mobile Phase are required. Automation makes the techniques more complicated & expensive. Advantages and disadvantages:

66. Thank you